The prototypical store-operated calcium-influx pathway ICRAC (for "Ca2+-release activated Ca2+ current") was identified in 1992. Since then, substantial information has been acquired about ICRAC's physiological and clinical importance, however, its molecular composition has remained elusive. Only recently did break-through findings identify two proteins that are essential in store-operated Ca2+ influx, namely stromal interaction molecule (STIM1) and CRAC Modulator 1 ((CRACM1) or Orai1). The combined overexpression of STIM1 and CRACM1 greatly amplifies store-operated currents and these possess the most defining characteristics of ICRAC. Subsequently, it was demonstrated that CRACM1 is a pore-forming subunit of the CRAC channel. Our preliminary data further suggest that the CRACM1 homologs CRACM2 and CRACM3 also form store-operated channels with distinct properties and that STIM2 can transiently activate CRACM proteins in store-dependent and store-independent manners. We therefore hypothesize that CRACM and STIM homologs represent a group of proteins that mediate store-operated Ca2+ entry with distinct functional properties. Specific Aim 1 tests the hypothesis that specific structural features of STIM1 and CRACM1 determine their function and interaction. We will assess the properties of the three CRACM homologs by modifying molecular sites that have been identified as possible structural determinants of specific, known functional characteristics of ICRAC. The CRACM mutant constructs will be co-expressed with STIM1 in HEK293 cells and functionally analyzed using biophysical approaches, including whole-cell patch-clamp and calcium imaging. In a next step, the molecular coupling elements of STIM1/2 and CRACM1/2/3 that result in CRAC channel activation will be investigated. The proposed CRACM mutant constructs will be co-expressed with wt STIM1/2 in HEK293 cells and STIM1/2 mutants will be co-expressed with wt CRACM1/2/3. Specific Aim 2 tests the hypothesis that STIM1 and STIM2, as well as CRACM1, CRACM2 and CRACM3 are alternate molecular components of the CRAC channel conferring different channel characteristics and Ca2+ signals in native cell systems. To this end, the human cell systems Jurkat T, HEK293, and HeLa cells will be used to correlate store-operated Ca2+ channel complements with agonist-mediated Ca2+ signals. Furthermore, cellular (DT40) and animal knock-out models (CRACM and STIM KO mice) will be used to assess CRACM and STIM complement and function. The proposed work will further our understanding of the molecular definition of CRAC calcium channels, greatly improving the prospects for developing therapeutic strategies involving store-operated Ca2+ influx in allergy, inflammation and autoimmune diseases.